Achieving high level, reproducible, stable transgene expression in mammalian cells remains a major bottleneck to critical areas of biomedical research, including production of protein biopharmaceuticals, gene therapy, cellular reprogramming, tissue engineering, as well as basic research into fundamental molecular and cellular biology processes and mechanisms of disease. The rationale for the proposed research is to develop the optimal components for a single and multiple transgene BAC expression system that will overcome long- standing problems in mammalian transgene expression and find applications over a wide range of biomedical research areas. Our long-term goal is to overcome existing problems in mammalian transgene expression in order to achieve the ability to engineer entire synthetic gene networks into human cells for improved ex vivo gene therapy and tissue engineering applications. The specific aims of this proposal are to: 1. Identify appropriate DNA genomic regions, cloned within BACs, and promoters that can be used to drive copy number dependent, position independent expression of single and multiple transgenes. 2. Optimize BAC / promoter combinations through the minimization of BAC size and addition / deletion of appropriate cis regulatory regions. 3. Apply this technology to specific test "driver" applications requiring multi-gene expression, including improved methods for generating induced pluripotent stem cells and facilitated high-throughput screening for chemicals which modulate stem cell pluripotency and differentiation. We propose to develop a general methodology enabling the engineering in a single step any mammalian cell line to express stably any single protein, or set of multiple proteins, at levels comparable to 100s fold higher than endogenous genes. Completion of our proposal should result in an improved methodology for generation of iPS cells and transdifferentiation, and more broadly a new methodology for tissue engineering applications. Our approach is innovative because it builds on special insights derived from our basic science investigations into how 10 and 30 nm chromatin fibers fold into interphase chromosomes, and the relationship between this "large-scale chromatin structure" and transcriptional activation. PUBLIC HEALTH RELEVANCE: This project is relevant to NIH's mission because it directly addresses the critical problem of expressing foreign genes in mammalian cells. This problem currently is a major technical bottleneck limiting progress in multiple applied areas including production of protein biopharmaceuticals, gene therapy, and stem cell engineering.